1. Field of the Invention
There is a continually expanding need to determine the presence of minute quantities of organic materials. Concentrations of interest range from about 10.sup.-4 to 10.sup.-12 M or even lower. Areas where the determinations are significant include the presence of drugs of abuse in physiological media, the metering of therapeutic dosages of drugs, disease diagnosis, where the presence, absence or amount of a particular organic material is relevant to the diagnosis of the disease and assaying for trace components in food. Other areas which are not of physiological interest include scientific investigation and assaying for trace contaminants in water or other fluids, quality control, and the like.
One approach to assays for specific materials comes under the class of immunoassays. Immunoassays employ a receptor, normally an antibody, which recognizes a specific spatial structure and charge distribution--epitope--in an organic molecule. The antibodies are relatively large molecules, of 150,000 or greater molecular weight and are protein in nature. Therefore, with most organic compounds of interest, the binding of the antibody to the organic compound provides significant enhancement in molecular weight, as well as a change in the environment of the organic compound, as compared to the solvent environment. In immunoassays, aqueous solvents are normally employed.
In radioimmunoassay, the great enhancement in molecular weight allows for separation of an organic compound which is bound to antibody and unbound organic compound. By having a detector molecule which is radioactive, one can determine the distribution of the radioactive compound between bound and unbound. This distribution is related to the concentration of the organic compound present in the unknown.
A second technique is a spin immunoassay technique, supplied by Syva Company, under the trademark FRAT.RTM.. In this technique, a stable free radical compound is bound to a compound resembling the unknown organic compound. The rate at which the spin label compound tumbles in solution affects the height of the electron spin resonance spectrum. When the spin label compound is bound to antibody, the rate is substantially slower than the unbound compound. By relating the peak height of the electron spin resonance spectrum to known standards, one can determine the amount of the unknown compound present in the solution.
Another technique uses an enzyme as the detector. In this technique, an enzyme is bound to the unknown compound and which is sold by Syva Company, under the trademark EMIT.RTM.. When the enzyme-bound compound is bound to an antibody, there is a substantial reduction in the enzyme activity. Therefore, by metering the enzyme activity, and relating enzyme activity to a standard, one can determine the amount of unknown in a solution.
2. Description of the Prior Art
U.S. Pat. No. 3,709,868 is exemplary of a radioimmunoassay. U.S. Pat. No. 3,960,834 is exemplary of a spin immunoassay. U.S. Pat. No. 3,654,090 and German Auslegungsschrift No. 2,223,385 are exemplary of enzyme immunoassays. Articles of interest include an article by Ludwig Brand and James R. Gohlke, Annual Review of Biochemistry, 41, 843-868 (1972) and Stryer, Science, 162, 526 (1968). Smith, FEBS Letters 77, 25, (1977) describes a fluorescent immunoassay, where thyroxine is bound to a fluorescer and quenches the fluorescer, the quenching being reversed by binding of antibody to thyroxine. See also, Ullman et al, J. Bio. Chem. 251, 4172 (1976).